Drug Resistance in HCV NS3/4A and Beyond - Inhibitor Binding versus Substrate Recognition In the last 3-5 years the treatment of Hepatitis C Virus (HCV) has been revolutionize by direct- acting antivirals (DAAs). NS3/4A protease was the first target for HCV where DAAs were developed. NS3/4A protease inhibitors (PIs) continue to be key components in the development of therapeutic options including combination therapies currently in phase 3 clinical trials. While the current PIs are highly potent against wil-type genotype 1 HCV, the rapid evolution of HCV and genotypic differences around the world allow viable variants with often multiple mutations that are resistant against these PIs, and decrease their effectiveness. Drug resistance is caused by a change in the balance of molecular recognition events that selectively weakens inhibitor binding but maintains the biological function of the therapeutic target. Disrupting the therapeutic target's activity is necessary but nt sufficient for avoiding resistance. We hypothesize that the impact of multiple mutations selected under the pressure of PI therapy or in different genotypes is not simply additive, but these mutations have interdependent effects causing structural and dynamic changes to confer resistance. We will elucidate the molecular mechanisms by which resistance occurs in HCV NS3/4A, and develop and implement strategies to design new inhibitors less susceptible to resistance. One key strategy is to compare inhibitor binding to substrate recognition and avoid functionally unessential contacts that are vulnerable to resistance mutations. The strategies we develop to design potent inhibitors less susceptible to resistance will also be translated to flaviviral proteases such as dengue, where no current DAA or vaccine exists.